Advances in molecular genetic techniques are revealing new details of the neuroanatomical organization of brain circuitry and the functional role of these circuits in behavior. Engineered viral vector constructs have been developed to label axonal projections of targeted neurons with unprecedented clarity, while others allow for retrograde trans-synaptic labeling of neurons providing inputs or anterograde trans-synaptic labeling of post-synaptic targets of axonal projections. Development of optogenetic and DREADD techniques provide the ability to functionally manipulate neural circuits to study their role in behavior while calcium indicators provide the ability to analyze the physiologic activity in targeted neuron populations. Together these approaches provide new insights into the functional organization of neural circuits. For example, optogenetic studies, using light activation of Channel Rhodopsin (ChR), have demonstrated the ability to functionally manipulate specific neural pathways to determine their role in behaviors including fear memory, anxiety, feeding, and movement. The analytic potential of these approaches is enhanced by the ability to target specific neuron populations, which are defined components of neural circuits. One approach involves the use of transgenic Cre-driver mouse lines in which Cre-recombinase is expressed under the control of gene-specific promoters. In recent years we characterized BAC-Cre driver lines from the GENSAT project that allow for targeting components of the neural circuits of the cerebral cortex and basal ganglia (Gerfen et al., 2013). Of particular significance, lines were characterized with selective labeling in cortical layer 2/3, in layer 4, in layer 5 and in layer 6. Each of these cortical layers contain neuron subtypes with distinct axonal projection patterns. These BAC-Cre lines provide unprecedented ability to study the specific function of these cortical subtypes in behavior. In the past year we collaborated with investigators in NIMH and NEI, at the Howard Hughes Medical Institute Janelia Farms Research, and the University of Pittsburgh in studies that used the BAC-Cre lines to determine the relationship between the organization of information transfer between sensory, motor and association cortical areas and the planning and initiation of movements. During this year neuroanatomical techniques were developed to analyze the functional organization of the relationship between the cerebral cortex and basal ganglia. Our work focuses on the organization of neural circuits responsible for integrating different modes of sensory and experiential information that is utilized in the planning and execution of behavior. To do this we use viral vectors that label the axons of specific cortical neuron subtypes in GENSAT BAC-Cre mouse lines. Viral vectors injected into different cortical areas in BAC-Cre lines expressing in cortical layers 2/3 and 5a map the connections between functional cortical areas, which are responsible for integration of information, while injections in BAC-Cre lines expressing in layers 5b and 6 label axonal projections to subcortical systems, which are involved in turning cortical activity into behavior. The labeled axons are visualized using immunohistochemical techniques in 50m sections of the brain and imaged to reveal multiple injections in different cortical areas reveal specific patterns of connectivity between functionally distinct cortical areas and subcortical systems. To analyze these complex patterns of connectivity we developed an efficient process for reconstructing the images through the whole mouse brain using the NIH ImageJ program (Paletzki and Gerfen, 2015). These fully reconstructed whole mouse brain image sets displaying the axonal projections of specific neuron subtypes in multiple cortical areas are registered to a common mouse brain atlas. The ability to register patterns of axonal projections obtained from many brains provides the ability to analyze the complex organization of the neural circuits integrating information between functional cortical areas and how it is transmitted to subcortical circuits responsible for behavior (Eastwood et al., 2019; Tappan et al., 2019). Work with the Janelia MouseLight Project provides an exciting advance in analysis of the connectivity of cortical neural circuits (Economo et al., 2016; Gerfen et al., 2017). The MouseLight Project has developed a platform for tracing the axonal projections of individual cortical neurons through the whole brain. Ongoing studies of the MouseLight project have analyzed the organization of reciprocal connections between the motor cortex and the thalamus at the single neuron level to reveal distinct subtypes of both cortical and thalamic neurons with specific patterns of axonal arborization ( Winnubst et al., 2019) In a collaborative study with the Svoboda lab at Janelia Research Campus, data from mapping projections of both populations and single layer 5 cortical pyramidal neurons in promotor cortical areas identified 2 subtypes based on their selective axonal projections to either thalamus or brainstem motor nuclei (Economo et al, 2018). Single cell mRNA expression profiling demonstrated specific genetic differences between these subtypes. In addition, behavioral studies combined with optogenetic stimulation and activity recording showed that the thalamic projecting subtype is involved in the preparatory activity (Guo et al., 2017), preceding goal directed movements, which engage the brainstem projecting cortical neuron subtype. In a collaboration with the Krauzlis lab in NEI, the role of the striatum in perceptual decision-making as distinguished from action selection was studied (Wang et al., 2018). Using a visual orientation-change detection task combined with selective optogenetic manipulation of either the direct or indirect striatal pathways it was demonstrated that activation of the direct pathway significantly increased the response to the sensory stimulus and not due to a general increase in response initiation. These results indicate a causal link between the activity in the direct pathway and decision-making dependent on perceptual bias. With Bryan Hooks at the University of Pittsburgh we studied the organization of cortical inputs to the striatum (Hooks et al., 2018). Cortico-striatal inputs are organized topographically generally with projections distributed over an area within the striatum such that there is convergence of inputs. This convergence integrates information from different cortical areas, such as from sensory and motor areas. In our study we used Cre-driver lines combined with injections of viral vectors that label axonal projections to map the projections from subtypes of layer 5 cortical neurons into the striatum. Data demonstrated that motor and sensory areas that are inter-connected project to overlapping regions in the striatum. For example, the upper limb area of the somatosensory cortex projects to a discrete part of the motor cortex, which projects back to the upper limb area of the somatosensory cortex and both of these cortical areas project to overlapping area of the striatum. The precision of the topographic organization of cortico-striatal inputs decreases along a gradient from primary somatosensory cortex to primary and secondary cortical areas. These data provide new insights into how information from the cortex is processed through the basal ganglia to affect behavior. The analytic process used in this study was further developed to allow resesarchers to analyze changes in neuronal activity in behavioral models to identify the neural circuits responsible for mental and neurologic disorders (Eastwood et al., 2019; Tappan et al, 2019).